CN105420212B

CN105420212B - Novel mycobacterium tuberculosis extracellular nuclease and application thereof

Info

Publication number: CN105420212B
Application number: CN201510953549.7A
Authority: CN
Inventors: 刘思国; 陈利苹; 宋宁宁
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2015-12-15
Filing date: 2015-12-15
Publication date: 2020-02-14
Anticipated expiration: 2035-12-15
Also published as: CN105420212A

Abstract

The invention discloses a novel mycobacterium tuberculosis extracellular nuclease and application thereof. The invention proves that Rv0888 is an extracellular nuclease of mycobacterium tuberculosis. Based on sequence analysis, Rv0888 nuclease has no homology with known extracellular nucleases, indicating that Rv0888 is a novel extracellular nuclease. The activity of Rv0888 protein is exerted with the aid of divalent ions, with optimal temperature and pH of 41 ℃ and 6.5, respectively. The invention also provides the application of four traditional Chinese medicine monomers of oleuropein, 6-gingerol, psoralen B and acteoside in inhibiting the nuclease. Site-directed mutagenesis studies showed that residues H353, D387, and D438 catalyzed the activity of Rv 0888. In vivo infection experiments prove that the retention capacity of the recombinant mycobacterium smegmatis Rv0888NS/MS and Rv0888S/MS in the lung of a mouse is obviously higher than that of pMV262/MS, and the Rv0888NS/MS and Rv0888S/MS can generate obvious pathological changes in the lung of the mouse, which indicates that Rv0888 is necessary for mycobacterial infection and pathogenicity.

Description

Novel mycobacterium tuberculosis extracellular nuclease and application thereof

Technical Field

The invention relates to an extracellular nuclease and application thereof, in particular to a novel mycobacterium tuberculosis extracellular nuclease and application thereof, belonging to the technical field of biology.

Background

Many bacteria produce extracellular nucleases which play important roles in bacterial virulence, biofilm formation, use of extracellular DNA for nutrition and degradation of DNA in extracellular traps (NETs). Extracellular nucleases such as Shewanella, Serratia, Staphylococcus aureus are capable of degrading extracellular DNA to obtain its essential nutrients-a source of phosphorus, carbon and nitrogen. Recent studies by Seper have shown that wild type Vibrio cholerae is able to degrade the DNA content of NETs by its own two extracellular nucleases (Dns and Xds). The extracellular nucleases of staphylococcus aureus can also degrade NETs, thereby triggering caspase-3 mediated immune cell death. Whereas the extracellular nuclease of Streptococcus agalactiae (Gbs0661) is capable of attacking NETs and is essential for bacterial virulence. (NETs-neutrophil extracellular traps are an important part of host defense responses, which allow the immobilization of microorganisms and subsequent clarification). In glucose-deficient media, the important nucleic acids released by deoxyribonuclease (Nuc B) released during the sporulation phase of bacillus subtilis 168, which are capable of degrading biofilms, are utilized by themselves.

Mycobacterium tuberculosis (m. tuberculosis) is a gram-positive bacterium, which is a pathogenic bacterium causing tuberculosis. Tuberculosis has been one of the highest mortality diseases in the world. In 2013, about 900 million people infected with tuberculosis and about 150 million people died from the disease, of which 36 million people were HIV positive. After entering the lung, M.tuberculosis is phagocytosed by macrophages and dendritic cells, however M.tuberculosis can spread among these immune cells and even spread infection by escaping these phagocytes from migrating to lymph nodes.

Extracellular nucleases of gram-positive bacteria play a very important role in the virulence of the bacteria and in the degradation of the DNA of the extracellular trap (released by neutrophils or macrophages). Previous studies on extracellular nucleases have focused mainly on Streptococcus agalactiae, Streptococcus pyogenes and Staphylococcus aureus. Although previous reports describe M.tuberculosis intracellular nucleases, to date no data has shown the presence of extracellular nucleases in M.tuberculosis. Next, the present invention for the first time found and confirmed that Rv0888 protein is an extracellular nuclease of mycobacterium tuberculosis.

Disclosure of Invention

An object of the present invention is to provide a novel mycobacterium tuberculosis extracellular nuclease and use thereof.

The second purpose of the present invention is to provide an inhibitor capable of inhibiting the extracellular nuclease of Mycobacterium tuberculosis.

In order to achieve the purpose, the invention adopts the following technical means:

in the present invention, we confirmed that the Rv0888 protein is the first extracellular nuclease from Mycobacterium tuberculosis and belongs to endonuclease/eThe xonuclease/phosphatase family (Pfam family PF03372) showed sphingomyelinase activity in the culture filtrate of M.tuberculosis. Further, the present invention also demonstrates that the extracellular nuclease has high activity in degrading various types of nucleic acids (genomic DNA, double-stranded PCR products, plasmid DNA and baker's yeast RNA), and that the assistance of divalent ions (Ca) is required for Rv0888 to exert its nuclease activity²⁺And Mn²⁺). The other four Chinese medicinal monomers (6-gingerol, acteoside, oleuropein, and psoralen B) can inhibit the nuclease activity of Rv 0888.

On the basis, the invention provides a novel mycobacterium tuberculosis extracellular nuclease which is named as Rv0888, and the amino acid sequence of the mycobacterium tuberculosis extracellular nuclease is shown as SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO. 5.

Furthermore, the invention also provides a nucleotide sequence for coding the mycobacterium tuberculosis extracellular nuclease. Preferably, the nucleotide sequence is shown as SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO. 6.

Expression vectors containing said nucleotide sequences and host cells containing the expression vectors naturally also fall within the scope of protection of the present invention.

Still further, the invention also provides the application of the mycobacterium tuberculosis extracellular nuclease in degrading nucleic acid.

Wherein, preferably, the nucleic acid comprises linear double-stranded DNA, circular plasmid DNA, chromosomal DNA or RNA.

Furthermore, the invention provides the application of oleuropein, 6-gingerol, psoralen B or acteoside in inhibiting the mycobacterium tuberculosis extracellular nuclease.

The Rv0888 nuclease activity was shown to be in the temperature range of 33 ℃ to 51 ℃ with an optimal temperature of 41 ℃. This temperature is consistent with the elevated body temperature of humans and animals following infection with mycobacterium tuberculosis, suggesting that Rv0888 may be involved in the virulence of mycobacterium tuberculosis. Similar to other deoxyribozymes/nucleases, Rv0888 nuclease also requires divalent ions for activity. The most preferred divalent ion is Mn²⁺And Ca²⁺And othersDivalent ion (Mg)²⁺,Ba²⁺,Ni²⁺) There was also a different degree of promotion (table 1). These results indicate that, as with previously reported E.coli nucleases, the catalytically active site of Rv0888 is not related to the diameter of the divalent ion, but rather the binding of these ions to different amino acids maintains their activity. The strong inhibitory effect of EDTA, a metal chelator, on Rv0888 activity suggests that metal ions are essential for nucleic acid activity and play a very important role in enzyme structural stability.

Amino acid substitution studies indicate that the amino acid at position D438 plays a key role in Rv0888 nuclease activity, and H353 and D387 also play a more positive role in Rv0888 activity. These important amino acid residues are different from other streptococcus pneumoniae ectonucleases EndA22 containing DRGH conserved motifs and from DKGH streptococcus agalactiae ectonucleases Gbs 06616. Rv0888 protein does not have any homology to other known nucleases (figure 9), so our data indicate that Rv0888 is one of the non-specific nuclease family members.

Tuberculosis is difficult to control due to its co-infection with HIV and the emerging untreatable or widely drug resistant mycobacterium tuberculosis. There is an urgent need to develop new drugs and more effective vaccines. There is therefore a need to understand the virulence and pathogenicity of mycobacterium tuberculosis on a genetic basis. Interestingly, four Chinese medicinal monomers (oleuropein, acteoside, 6-gingerol and psoralen B) can inhibit activity of Rv 0888. Previous studies on inhibitors have focused mainly on small molecule inhibitors, mainly factors related to protein and pathogen biosynthesis: for example, structural homolog ML141 prevents s.aureus from entering endothelial cells; cystatin K11777 blocks the entry of coronaviruses and filoviruses into the host cell; compound 1771 blocks phosphatidylglycerol-LtaS binding and inhibits LTA synthesis during ItaS expression in s.aureus and e.coli; BAS00127538 was used to inhibit the passage of acinetobacter baumannii Lipid II, a precursor of cell wall biosynthesis. Subsequent drug studies have shifted to cell wall proteins and proteases: sortase inhibitors can be used to treat nosocomial infections with staphylococcus aureus without the side effects of standard antibiotics; orlistat, an inhibitor of mycobacterium tuberculosis phospholipase/thioesterase (Rv3802 c); an inhibitor of virulence factor mPTPB of tuberculosis obtained by organic catalysis; six small molecules were shown to inhibit streptococcus pneumoniae surface nuclease EndA 31; importantly, the traditional Chinese medicine monomer is reported to be capable of effectively inhibiting nuclease of bacteria for the first time. At present, the research on new drugs for pathogens with wide drug resistance gradually turns to traditional Chinese medicines. Therefore, the verification of new virulence factors and the screening of traditional Chinese medicine inhibitors are probably beneficial to the discovery of new drugs for treating tuberculosis.

In vivo infection experiments show that recombinant Mycobacterium smegmatis Rv0888NS/MS and Rv0888S/MS continue to be retained in the lung when the mouse is infected with 17d, and histopathological analysis shows that the lung of the mouse is obviously changed pathologically when the mouse is infected with 7d, which means that Rv0888 is probably a virulence factor of the Mycobacterium tuberculosis and is probably related to the retention of the Mycobacterium tuberculosis in the lung of a host. Previous studies have reported the localization of different bacterial nucleases in bacteria: streptococcus pyogenes are secreted into the culture supernatant, the nucleases of Streptococcus pneumoniae are localized on the membrane and Streptococcus pyogenes, and Streptococcus suis is localized on peptidoglycan. Mycobacterium tuberculosis Rv0888 contains a signal peptide and can be secreted into the culture medium. This secreted form is more effective in the pathogenicity of M.tuberculosis than membrane-localized nucleases like Streptococcus pneumoniae EndA, because they have easier access to their substrates. The properties of Rv0888 nuclease in this study made it easier for us to understand the pathogenicity of mycobacterium tuberculosis and perhaps helped us to obtain new drugs for tuberculosis treatment.

Drawings

FIG. 1 shows the purification of recombinant Rv0888 protein and mass spectrometric verification;

(A) SDS-PAGE analysis of affinity purification of Rv0888 protein 20mM Tris-HCl-150mM NaCl-10% glycerol containing a concentration gradient of imidazole eluted Rv0888 protein. 1:500mM imidazole; 2:200mM imidazole; 3:100mM imidazole; 4:80mM imidazole; 5:70mM imidazole; 6:60mM imidazole; 7:40mM imidazole; 8:20mM imidazole; m: protein molecular mass Marker. (B) And purifying the Rv0888 protein by ion exchange chromatography. (C) And purifying the Rv0888 protein by gel filtration chromatography. (D) And analyzing the peptide mass fingerprint spectrum of the ion line time mass spectrum by the Rv0888 protein through matrix-assisted laser. Sequence in-box consistent with Rv0888 protein matching.

FIG. 2 is a schematic representation of the digestion of different nucleic acids by purified Rv0888 protein;

the reaction conditions are as follows: 20mM Tris-HCl pH 7.5and 5mM MgCl237 ℃ for 1 hour. (A) Purified Rv0888 protein digests different DNAs. M is DL5000DNA Marker; 1 chromosomal DNA with 20mM Tris-HCl (pH 7.5); chromosomal DNA and purified Rv 0888; 3 circular plasmid DNA with 20mM Tris-HCl (pH 7.5); 4, circular plasmid DNA and purified Rv 0888; 5 Linear double-stranded DNA with 20mM Tris-HCl (pH 7.5); linear double-stranded DNA and purified Rv 0888; (B) purified Rv0888 protein digests RNA. M is DL5000DNA Marker; 1, baker's yeast RNA and 20mM Tris-HCl (pH 7.5); (ii) a2, bread yeast RNA and purified Rv 0888; (C) the deoxyribonuclease activity requires a cation. M is DL5000DNA Marker; 1 circular plasmid DNA with 20mM Tris-HCl (pH 7.5); 2 circular plasmid DNA and purified Rv0888 containing 5mM CaCl₂And 5mM MnCl₂(ii) a Circular plasmid DNA and purified Rv0888 containing 5mM CaCl₂，5mM MnCl₂And 20mM EDTA.

FIG. 3 is the effect of pH or temperature on the activity of Rv0888 enzyme;

(A) the effect of temperature on Rv0888 enzyme activity was analyzed by agarose gel electrophoresis. The reaction conditions are as follows: 20mM Tris-HCl pH 6.5,5mM CaCl₂and 5mM MnCl₂Incubate for 1 h. M is DL5000DNA Marker; 1: circular plasmid DNA; 2-11 circular plasmid DNA and Rv0888 protein at 33-51 deg.c and 2 deg.c interval. (B) The deoxyribonuclease activity was quantified spectrophotometrically, with error bars representing standard deviations. (C) The effect of pH on Rv0888 enzyme activity was analyzed by agarose gel electrophoresis. The reaction conditions are as follows: 20mM Tris-HCl,5mM CaCl₂and 5mM MnCl₂Incubate at 37 ℃ for 1 h. M is DL5000DNA Marker; 1,3,5,7,9, circular plasmid DNA-pH 6.0-pH8.0, interval 0.5; 2,4,6,8,10 circular plasmid DNA and Rv0888 protein-pH6.0-pH8.0, spaced 0.5 apart. (D) The deoxyribonuclease activity was quantified spectrophotometrically, with error bars representing standard deviations.

FIG. 4 is a Michaelis kinetic assay of deoxyribonuclease (A) and ribonuclease (B) activities of Rv 0888;

the reaction conditions are as follows: 20mM Tris-HCl pH 6.5,5mM CaCl₂and 5mM MnCl₂Incubate at 41 ℃ for 1 h. K for DNA and RNA_mThe values are respectively: 0.306 plus or minus 0.04mg/mL and 0.012 plus or minus 0.01 mg/mL; DNase and RNase V_maxThe values are respectively: 600.56U/mg/min and 241.11U/mg/min.

FIG. 5 shows oleuropein, 6-gingerol, acteoside, psoralen B inhibits Rv0888 from degrading circular plasmid DNA;

the reaction conditions are as follows: 20mM Tris-HCl pH 6.5,5mM CaCl₂and 5mM MnCl₂Incubate at 41 ℃ for 1 h. (A) M is DL5000DNA Marker; 1: circular plasmid DNA; 2, circular plasmid DNA and Rv 0888; 3, circular plasmid DNA and Rv0888 contain 50% ethanol; circular plasmid DNA and Rv0888 contained 50% DMSO; 5-7 circular plasmid DNA, Rv0888 and oleuropein (0.5 mM,1mM,2mM, respectively); 8-10 circular plasmid DNA, Rv0888 and 6-gingerol (0.5 mM,1mM,2mM, respectively); 11-13 circular plasmid DNA, Rv0888 and psoralen B (0.5 mM,1mM,2mM, respectively); 14-16 circular plasmid DNA, Rv0888 and acteoside (0.5 mM,1mM,2mM, respectively) (B) spectrophotometrically to quantify the deoxyribonuclease activity, error bars represent standard deviations.

FIG. 6 is a Rv0888 site-directed mutagenesis;

10 amino acid residues are replaced by site-directed mutagenesis of the gene: N131A, E267A, G303A, H353A, D387A, N389A, D438A, D472A, H473A, D472A-H473A. (A) Multiple sequence alignments of the Rv0888 protein with other members of the endonuclease/exonuclease/phosphatase family. Query is Rv 0888; # conserved residues. (B) Relative activity of 10 protein mutants to Rv0888 protein. The experimental reaction conditions were: 20mM Tris-HCl pH 6.5,5mM CaCl₂and 5mM MnCl₂Incubate at 41 ℃ for 1 h. Error bars represent standard deviation.

FIG. 7 shows the retention of recombinant Mycobacterium smegmatis in the lungs of mice;

recombinant Mycobacterium smegmatis pMV262/MS, Rv0888NS/MS and Rv0888S/MS nasal drops infected BALB/c mice, bacterial load in the lungs at 4h,24h,4d,7d, and 17d infection.

FIG. 8 is a histopathological analysis of mouse lungs;

recombinant Mycobacterium smegmatis pMV262/MS, Rv0888NS/MS and Rv0888S/MS were nasally infected with BALB/c mice 7d, PBS was used as a negative control. (A) PBS control; has no pathological changes. (B) Pmv 262/MS; no pathological changes. (C) Rv0888 NS/MS; mild alveolar epithelial cell proliferation. (D) Rv 0888S/MS; mild bleeding and alveolar epithelial cell proliferation.

Fig. 9 is a multiple sequence alignment of Rv0888 with other bacterial extracellular nucleases.

The sequences of Rv0888, s.pyogenes Sda 1(AAS09918), l.lactis YbfB (YP — 001031529), s.pneumoniae EndA (CAA38134), s.agalactiae NucA (NP — 735111) were analyzed by MultAlin software alignment.

Detailed Description

The invention will be further described with reference to specific embodiments and drawings, the advantages and features of which will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 cloning and Effect verification of Mycobacterium tuberculosis extracellular nuclease Rv0888

Materials and methods

1.1 animals, strains and growth conditions

Mice were purchased from Beijing Witonglihua, and all animals used in the experiments followed the animal welfare and ethics of the animal ethics Committee of Heilongjiang province, the science and technology institute of Heilongjiang province, the people's republic of China, and were approved and supervised by the animal welfare Committee of the Harbin veterinary institute. All experiments used the smallest number of mice possible and minimized the pain of the animals as much as possible.

Mycobacterium smegmatis (m.smegmatis) was cultured in Middlebrook 7H9(BD Biosciences) containing 10% OADC (oleic acid/albumin/catalaseen, BD Biosciences), 0.05% Tween-80(Amresco), and 0.2% glycerol (Sigma-Aldrich) e.coli DH5 α strain was used for plasmid preparation, e.coli Rosetta 2 strain was used for protein expression, all e.coli (e.coli) were cultured in LB culture.

1.2 cloning and expression

The amino acid sequence of Rv0888 (shown in SEQ ID NO. 1) was predicted by using an online service SignalP 4.0server (http:// www.cbs.dtu.dk/services/SignalP /), and Rv0888 (shown in SEQ ID NO. 6), Rv0888NS (shown in SEQ ID NO. 4) and Rv08 0888S genes (shown in SEQ ID NO. 2) were amplified using the primers in Table 1, using the M.tuberculosis H37Rv genome as a template. PrimeSTAR MaxDNA (TaKaRa Bio) polymerase was used for PCR amplification.

TABLE 1 primers for use with the Mycobacterium tuberculosis Rv0888 Gene

The Rv0888 amplicon was ligated into the pET-22b vector (Novagen) containing a 6 histidine tag at the C-terminus, followed by transformation of Rosetta 2(DE3) E.coli with the recombinant plasmid pET-22b-Rv 0888. Rv0888 protein was expressed as inclusion bodies. Recombinant Rosetta 2(DE3) E.coli cultured in LB medium (containing 200. mu.g/mL of ampicillin and 34. mu.g/mL of chloramphenicol) at 37 ℃ OD600 to 0.6-1.0, followed by induction with 1mM IPTG at 37 ℃ for 4 h. The Rv0888NS and Rv0888S amplicons are connected to the pMV262 vector, and the recombinant plasmids pMV262-Rv0888NS and pMV262-Rv0888S are transfected into Mycobacterium smegmatis (M²155). Recombinant Mycobacterium smegmatis (rMS) was cultured at 37 ℃ in 7H9 medium (containing 0.05% Tween-80, 0.2% glycerol, 10% OADC, 50. mu.g/mL kanamycin) to OD600 to 0.8-1.0. The expression of the protein was analyzed by SDS-PAGE.

1.3 site-directed mutagenesis

PCR amplification using PrimeSTAR MaxDNA polymerase ((TaKaRa Bio)) using primers complementary to the mutations (Table 2), recombinant plasmid pET-22b-Rv0888 as template, yielded 10 mutants (N131A, E267A, G303A, H353A, D387A, N389A, D438A, D472A, H473A, D472A-H473A). PCR products were digested with DI pn, disrupted methylated DNA. the mutated plasmids transformed DH5 α competent cells.

TABLE 2 complementary mutant nucleotides used herein

1.4Rv0888 protein purification

Collect 4L Rosetta 2(DE3) E.coli bacterial culture containing pET-22b-Rv0888, resuspend pellet with 200mL buffer A (20mM Tris-HCl,150mM NaCl, 10% glycerol, pH8.0), crush with high pressure crusher (Constant system USA,30kpsi) at 4 ℃ and centrifuge at 1500rpm for 30min at 4 ℃ to collect pellet. The pellet was sonicated (350W,3s/3s,4 ℃) for 10min using buffer A resuspension, and the pellet was collected by centrifugation at 1500rpm for 30min at 4 ℃. The pellet was resuspended in 80mL of 2M urea, stirred at 4 ℃ for 4h, centrifuged at 4 ℃ at 1500rpm for 30min and the pellet collected. The pellet was resuspended in 80mLbuffer B (20mM Tris-HCl,150mM NaCl,6M urea, 20% glycerol, pH8.0), solubilized at 4 ℃ overnight, centrifuged at 1500rpm at 4 ℃ for 30min, and the pellet was discarded leaving the supernatant.

The supernatant was dialyzed against 2L buffer C (20mM Tris-HCl,150mM NaCl,5M urea, 20% glycerol, pH8.0) and the dialysate was changed every 12h, urea was reduced by 1M and the other components were unchanged until no urea was present. The dialyzed protein was centrifuged at 15000rpm at 4 ℃ for 30 min. The supernatant was passed through a Ni Sepharose 6Fast Flow resin (GEHealthcare) purification column, and unbound protein was washed with 60mL buffer E (20mM Tris-HCl,500mM NaCl, 10% glycerol,20mM MIDazole, pH8.0), 60mL buffer F (20mM Tris-HCl,500mM NaCl, 10% glycerol,40mM MIDazole, pH8.0) and 100mL buffer G (20mM Tris-HCl,1M NaCl, pH 8.0). The target protein was eluted with 5mL buffer H (20mM Tris-HCl,150mM NaCl, 10% glycerol 300mM imidazole, pH8.0) and 5mL buffer I (20mM Tris-HCl,150mM NaCl, 10% glycerol,500mM imidazole, pH 8.0). The collected proteins were purified by SDS-PAGE.

The affinity chromatography purified protein was concentrated to 2mL using a 30kDa ultrafiltration tube (Millipore), and the concentrated protein was passed through a HiLoad 16/600Superdex 200pg column (GEHealthcare) equilibrated with buffer J (20mM Tris-HCl, pH8.0) in advance, and the protein under all peaks was collected and confirmed by SDS-PAGE analysis. The validated protein was concentrated to 2mL over a Resource S5 mL column (GE Healthcare) previously equilibrated with buffer J. The target protein was eluted linearly with 0M-2M NaCl. The collected proteins were verified by SDS-PAGE analysis.

1.5 Mass Spectrometry of Rv0888 protein

The purified protein was subjected to SDS-PAGE, and the gel containing the target protein was excised and cut into 1mm pieces³The left and right pieces were placed in a centrifuge tube and washed once with water, then destained for 15 minutes with 50% (v/v) ACN/25mM ammonium bicarbonate (100. mu.L, pH8.0) and repeated 3 times until the color was depleted, the gel pieces were washed 1 time with distilled water, dipped into 30. mu.L 100% ACN for 5 minutes, dehydrated, gel pieces whitened and then drained at room temperature, 8. mu.L Trypsin enzyme solution (0.1mg/mL) was added, left and right overnight at 37 ℃ for 16 hours, 0.3. mu.L of the digested sample was added to 0.3. mu.L of a matrix (5mg/mL α -cyclono-4-hydroxycarinamic-acid (Fluka) in 50% (v/v) acetotrionic acid (Fisher) and 0.1% (w/v) trionic acid (mass spectrometry (DIMA)) on the sample plate, air dried, and the mass spectrometry (Swission time with matrix assisted laser analysis) was used to verify the results using a secondary mass spectrometry (Swission analysis) as a search for the error of the protein fragment, 2. origin data, the analytical method, the error of the analytical method of protein analysis, the analytical method of 0.1. + -. S. 7. + -. S.

1.6Rv0888 nuclease substrates

To test the specificity of the recombinant nuclease Rv0888 for the substrate. 10 μ L of reaction system, reaction buffer: 20mM Tris-HCl,5mM MgCl₂pH 7.5, 0.2. mu.g of linear dsDNA (PCR product), circular plasmid DNA (pGEX-6p-1 plasmid), chromosomal DNA (E.coli DNA) or baker's yeast RNA (Sigma-Aldrich) was added to react with 3. mu.g of purified Rv0888 protein in a water bath at 37 ℃. Buffer (20mM Tris-HCl pH 7.5) as a negative control instead of Rv0888 protein. After 60min, 10mu.L of the reaction solution was analyzed by adding 1. mu.L of 10 × loading buffer running 1% nucleic acid gel electrophoresis. All experiments were repeated three times.

1.7 Effect of divalent ions and Metal chelators on Rv0888 Activity

Divalent ions are usually deoxyribonuclease or are required for nucleases. The effect of divalent ions on nuclease activity was determined as follows: rv0888 protein with 5mM different divalent ion salt (CaCl)₂,MgCl₂,MnCl₂,BaCl₂,NiCl₂) And different combinations of divalent ion salts (CaCl)₂+MgCl₂,CaCl₂+MnCl₂,MgCl₂+MnCl₂) The reaction was carried out at 37 ℃ for 1 h. Results were analyzed by 1% nucleic acid gel electrophoresis. All experiments were repeated three times.

1.8 Effect of temperature and pH on Rv0888 Activity

The effect of different pH on the activity of Rv0888 was examined using optimal divalent ions and 20mM Tris-HCl buffer (pH 6.0-pH8.0, interval 0.5) at 37 ℃ for 1h of reaction. The effect of different temperatures on Rv0888 activity was examined using optimal divalent ions and optimal pH in the temperature range 33 ℃ -51 ℃ (2 ℃ apart). All results were verified by 1% nucleic acid gel electrophoresis analysis. The activity of Rv0888 was quantified using a spectrophotometric method previously reported. 200 μ L of reaction system: 20 μ L of 10x reaction buffer (100mM Tris-HCl,50mM CaCl)₂,50mM MnCl₂pH 7.5); 20 μ L of circular plasmid DNA (100 ng/. mu.L of pGEX-6 p-1); 10 μ L of Rv0888 protein (1 mg/mL); 150 μ L of sterile water. The reaction was stopped by adding 8. mu.L of 0.5M EDTA after 1 hour at 37 ℃. The reaction mixture was diluted to 2mL with sterile water and used in a Nanophotometer^TMThe measurement by the PealUltramicro UV-Vis spectrophotometer was carried out at 260 nm. The amount of enzyme required to degrade the product of the substrate at 41 ℃ per minute to increase the absorbance at 260nm by 0.001 is defined as one enzyme activity unit (U).

1.9 kinetic study of the Rv0888 nuclease Activity

Calf thymus DNA and baker's yeast RNA are used as substrates, and parameters of the Rv0888 nuclease mie kinetics are measured by a spectrophotometry method. At 41 ℃ and pH 6.5Mu.g of purified Rv0888 protein was incubated with different concentrations of calf thymus DNA (0.00625mg/mL,0.0125mg/mL,0.025mg/mL,0.05mg/mL,0.1mg/mL,0.2mg/mL,0.4mg/mL,0.6mg/mL,0.8 mg/mL; RNA:0.00625mg/mL,0.0125mg/mL,0.025mg/mL,0.05mg/mL,0.1 mg/mL). 20mM Tris-HCl (pH 6.5) instead of Rv0888 protein served as a negative control. After 1h of reaction, 5% ice final strength perchloroacetic acid was added, followed by dilution to 2mL with sterile water. The diluted solution was measured with a Nanophotometer^TMThe measurement by the PealUltramicro UV-Vis spectrophotometer was carried out at 260 nm. All experiments were repeated three times.

1.10Rv0888 inhibitor screening

The inhibitors of Rv0888 are screened by using sodium danshensu, gastrodine, leonurine hydrochloride, salidroside dissolved in water, oleuropein, chlorogenic acid, eleutheroside, 6-gingerol, crocin, curcumin, resveratrol, acteoside, polydatin, echinacoside, geraniin, rutin, phloridzin, baicalin, naringin, myricetin, silymarin, cnidium lactone, gambogic acid, rhein, puerarin dissolved in 50% ethanol, rhoifolin, psoralen B, glaucophylline C, methoxykavain, gliquidone, pterostilbene, curcumol, chicoric acid, loureirin B, rosiglitazone hydrochloride, L-lithospermum 50% DMSO, α -mangostin, harpagoside, coniferyl ferulate dissolved in 50% methanol, 3 μ g of purified Rv 88 protein, different from 0.3mM at 37 ℃, and the reference method for testing the activity of the traditional Chinese medicines by incubating with 50% methanol and 50% ethanol or more.

1.11 pulmonary infections

SPF female BALB/c mice (Beijing Wittitonia) 5-6 weeks old were used for the pulmonary infection test. The recombinant Mycobacterium smegmatis containing pMV262, no signal peptide Rv0888NS-pMV262 or signal peptide Rv0888S-pMV262 respectively is cultured in 7H9 culture medium, and the thalli in the logarithmic growth phase are collected and washed twice with 2mL of PBS for standby. The mice were randomly divided into 4 groups of 15 mice each and were anesthetized with ether prior to infection. 50 μ L PBS resuspension 2X 10⁷cfu recombinant Mycobacterium smegmatis infected mice nasally, PBS as a negative control. After infection for 4h,24h,4d,7d, and 17dEach group sacrificed 3 mice. The lungs were removed under sterile conditions, the left lung was ground and spread on solid plates containing 50. mu.g/mL kanamycin LB, and incubated at 37 ℃ for 3-4 days for colony counting. Approximately 1/3 of the right lung was fixed in 10% formalin for histopathological analysis.

Each experiment was repeated three times. Data were analyzed using Prism software (version 5.0; GraphPad, San Diego, CA, USA) and a two-sided test was used to count the significance of the data. A P <0.05 was considered significant (. times.p < 0.001).

Two results

2.1 expression and purification of Rv0888

The SignalP service prediction result shows that amino acids 1-31 of the Rv0888 are signal peptide sequences, and the Rv0888 gene segment without the signal peptide sequences is cloned by using the H37Rv genome as a template. To facilitate purification of recombinant proteins with 6 × His-tagged expressed in E.coli, they were first purified by affinity chromatography (FIG. 1A). The eluted Rv0888 protein was passed through an ion exchange column (fig. 1B) and finally through a gel filtration chromatography column to obtain protein of over 98% purity (fig. 1C).

2.2Rv0888 protein Mass Spectrometry validation

The purified Rv0888 protein was analyzed by mass spectrometry and the peptide mass fingerprinting results submitted to Mascot, searching the SwissProt database. 10 results were returned, with a score of 78 from M.tuberculosis Rv0888 protein (GeneBank Accession No. NP-215403.1) (see FIG. 1D).

2.3 specificity of the Rv0888 nuclease Activity

To confirm the nuclease activity of Rv0888, the purified proteins were incubated with different nucleic acids (linear double-stranded DNA (PCR product), circular plasmid DNA (pGEX-6p-1 vector), chromosomal DNA (e.coli DNA) or baker's yeast RNA). The results show that Rv0888 protein is able to degrade all nucleic acid substrates (fig. 2A and 2B). These results show that Rv0888 is a non-specific nuclease.

2.4 Effect of divalent ion and Metal ion chelators on Rv0888 Activity

The test result of the influence of different divalent ions on the activity of the Rv0888 nuclease shows that under the condition of no divalent ions,no nuclease activity of Rv0888 was detected. The enzyme activity was in the presence of 5mM CaCl₂And 5mM MnCl₂The best is the case. Other salts of divalent ions (CaCl)₂,MgCl₂,BaCl₂and NiCl₂) Also shown are different promoting effects on Rv0888 nuclease activity, and 20mM EDTA was able to completely inhibit Rv0888 activity (see table 3; fig. 2C).

TABLE 3 Effect of divalent ions on Rv0888 Activity (error bars represent standard errors)

ND-not detected

2.5 Effect of pH and temperature on the Activity of Rv0888

The results of the effect of pH and temperature on Rv0888 activity (substrate is a circular plasmid) are shown in figures 3A and 3C, respectively. Rv0888 was active at pH 6.0-8.0, with the best activity at pH 6.5 (FIG. 3D). Rv0888 was most active at 41 ℃ in an extensive temperature test (fig. 3B).

2.6 kinetic study of the Rv0888 nuclease Activity

Determination of kinetic parameters of Rv0888 nuclease, with reference to the following conditions: the substrate is calf thymus DNA and baker's yeast RNA; at 41 deg.C, pH 6.5, contains 5mM of divalent ion salt (CaCl)₂And MnCl₂). Results of the Michaelis kinetic assay show K for DNA and RNA_mThe values were 0.306. + -. 0.04mg/mL and 0.012. + -. 0.01mg/mL, respectively (FIGS. 4A and B). The Vmax for DNase and RNase were 600.56U/mg/min and 241.11U/mg/min, respectively (FIGS. 4A and B).

2.7Rv0888 inhibitors

The results of inhibitor screening showed that four herbal monomers (oleuropein, 6-gingerol, psoralen B, and acteoside) were able to inhibit Rv0888 nuclease activity, with the inhibitory effect increasing with increasing concentration (fig. 5A and 5B).

2.8 site-directed mutagenesis

Multiple sequence alignment of Rv0888 with endonuclease/exonuclease/phosphatase family homologous proteins revealed 9 highly conserved amino acids (N131, E267, G303, H353, D387, N389, D438, D472 and H473) (fig. 6A). The catalytic active site of Rv0888 protein was verified by site directed mutagenesis experiments to alter specific codons to correspond to the amino acid residues.

Alanine was substituted for 9 conserved amino acids, resulting in 10 muteins (N131, E267, G303, H353, D387, N389, D438, D472, D473 and D472-D473). Alanine is often used in place of other amino acids because it lacks bulky side chains and therefore does not disrupt the backbone conformation. The results of comparing the relative activities of the 10 protein mutants with that of Rv0888 protein showed that D438A lost 92% of activity, H353A lost approximately 50% of activity, and H473A and D472A-H473A lost approximately 30% of activity. N131A, E267A, G303A, N389A, and D472A lost about 10%, 4%, 6%, 11%, and 15% activity, respectively (fig. 6B).

2.9 Retention and histopathological analysis of recombinant Mycobacterium smegmatis in the Lung

The lung is the entrance of a mycobacterium tuberculosis infection, which is capable of adhering to and invading the epithelial cells of the lung. And (3) carrying out overexpression of Rv0888 by using mycobacterium smegmatis, and detecting the pathogenicity and retention capacity of the recombinant mycobacterium smegmatis in the lung. Recombinant M.smegmatis pMV262/MS, Rv0888NS/MS and Rv0888S/MS were separately infected by nasal drops to mice. Bacterial load in the lungs was examined after 4h,24h,4d,7d, and 17d of infection (fig. 7). At all time points, there was no significant difference in bacterial load between the Rv0888NS/MS and the Rv0888S/MS groups, however, the bacterial load was significantly higher for the Rv0888NS/MS and the Rv0888S/MS groups than for the pMV262/MS group after infection at 4d,7d and 17 d. More importantly, after 17d infection, the lungs of the pMV262/MS group were almost completely cleared of Mycobacteria, while the Rv0888NS/MS and Rv0888S/MS groups also had some number of Mycobacteria present.

Histopathological analysis showed no pathological changes in the lungs of mice in the pMV262/MS group 7d after infection, whereas the lungs of mice in the Rv0888NS/MS group showed mild alveolar epithelial cell proliferation and the lungs of mice in the Rv0888S/MS group showed mild hemorrhage and alveolar epithelial cell proliferation (FIG. 8). The above results indicate that nuclease activity is essential in the protection of mycobacteria against pulmonary tissue clearance and may be associated with the pathogenicity of mycobacteria.

Claims

1. The application of acteoside in inhibiting mycobacterium tuberculosis extracellular nuclease is disclosed, wherein the amino acid sequence of the mycobacterium tuberculosis extracellular nuclease is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5.